The mammalian target of rapamycin (mTOR) pathway is considered a major regulator of cell growth (Guertin, D. A., and Sabatini, D. M. Cancer Cell 2007, 12, 9). The mTOR serine/threonine kinase is the founding component of the pathway and the catalytic subunit of two functionally distinct protein complexes, mTORC1 and mTORC2. mTORC1 contains the large protein Raptor, as well as mLST8/GβL and PRAS40, whereas mTORC2 is defined by the protein Rictor and also includes Sin1, Protor, and mLST8/GβL. Growth factors, such as insulin and IGF, activate both complexes, and they are important downstream effectors of the PI3K/PTEN signaling network. Additionally, the availability of nutrients, like amino acids and glucose, regulates mTORC1. Many insights into mTOR signaling have come from investigations into the mechanism of action of rapamycin, a bacterially produced macrolide inhibitor of mTOR that has diverse clinical applications as an anti-fungal, immunosuppressant, and anti—Cancer drug. Rapamycin acts through an unusual allosteric mechanism that requires binding to its intracellular receptor, FKBP12, for inhibition of its target. Under acute treatment, rapamycin is thought to selectively inhibit mTORC1, which is often referred to as the rapamycin—Sensitive complex. Conversely, mTORC2 is considered rapamycin-insensitive, although its assembly can be inhibited by prolonged rapamycin treatment in some cell types. Because of its perceived potency and selectivity, rapamycin is commonly used in research experiments as a test of the involvement of mTORC1 in a particular process.
Two downstream mTORC1 substrates that were identified, in part, by their sensitivity to rapamycin are the S6 kinases (S6K1 and S6K2) and the translational inhibitor 4E-BP1. Both proteins mediate important links between mTORC1 and the cell growth machinery, largely through their influence on cap-dependent translation. All nuclear-encoded mRNAs possess a 5′,7-methyl guanosine cap, which is recognized and bound by the small protein eIF-4E. Under growth-promoting conditions, eIF-4E also associates with the large scaffolding protein eIF-4G, the eIF-4A helicase, and the eIF-4B regulatory protein, together forming the eIF-4F complex. This complex, in conjunction with the eIF3 pre-initiation complex, delivers the mRNA to the 40 S ribosomal subunit and primes the translational apparatus. 4E-BP1 interferes with this process by binding to eIF-4E and preventing the formation of a functional eIF-4F complex. However, its ability to do this is blocked by phosphorylation at four sites, two of which are considered rapamycin—Sensitive. S6K1 also plays a role in regulating translational initiation by phosphorylating the S6 protein of the 40 S ribosomal subunit and by stimulating eIF-4A helicase activity.
Despite the connections of mTORC1 to the translational machinery, the effects of rapamycin on mammalian cell growth and proliferation are, oddly, less severe than its effects in yeast. In Saccharomyces cerevisiae, rapamycin treatment induces a starvation-like state that includes a severe G1/S cell cycle arrest and suppression of translation initiation to levels below 20% of non-treated cells. Moreover, in yeast rapamycin strongly promotes induction of autophagy (self-eating), a process by which cells consume cytoplasmic proteins, ribosomes, and organelles, such as mitochondria, to maintain a sufficient supply of amino acids and other nutrients. The effects of rapamycin in mammalian cells are similar to those in yeast, but typically much less dramatic and highly dependent on cell type. For instance, rapamycin only causes cell cycle arrest in a limited number of cell types and has modest effects on protein synthesis. Moreover, rapamycin is a relatively poor inducer of autophagy, and it is often used in combination with LY294002, an inhibitor of PI3K and mTOR. These inconsistent effects may explain why, despite high expectations, rapamycin has had only limited success as a clinical anti—Cancer therapeutic.
Recently, it was reported that highly potent and selective ATP—Competitive mTOR inhibitors, that directly inhibits both complexes, impair cell growth and proliferation to a far greater degree than rapamycin (Thoreen et al. J. Biological Chem. 2009, 284, 8023. Feldman et al. PLoS Biology 2009, 7, 731. Garcia-Martinez et al. Biochem. J. 2009, 421, 29. Yu et al. Cancer Res, 2010, 70, 621). These effects are independent of mTORC2 inhibition and are instead because of suppression of rapamycin-resistant functions of mTORC1 that are necessary for cap-dependent translation and suppression of autophagy. These effects are at least partly mediated by mTORC1-dependent and rapamycin-resistant phosphorylation of 4E-BP1. These findings challenge the assumption that rapamycin completely inhibits mTORC1 and indicate that direct inhibitors of mTORC1 kinase activity may be more successful than rapamycin at inhibiting tumors that depend on mTORC1.
Previously, we disclosed that 6-morpholin-4-yl-pyrazolo[3,4-d]pyrimidine derivatives (Formula I) are potent and selective PI3K/mTOR inhibitors (U.S. provisional application Ser. No. 61/199,019 and 61/214,828). In general, these compounds inhibit both PI3K (particularly the α, β, δ isoforms) and mTOR, displaying little or no selectivity among these kinases.

In this invention, we discovered that the 4-urea-phenyl substituted 6-morpholin-4-yl-pyrazolo[3,4-d]pyrimidine derivatives (Cy=4-urea-phenyl) are potent and selective mTOR kinase inhibitors with much less PI3K inhibitory activities. Giving the exciting anti-tumor activities, at least in pre—Clinical studies, of mTOR kinase inhibitors as discussed above, these novel compounds may also have potential in treating diseases mediated by mTOR such as cancer, immune disorders, cardiovascular disease, ocular disease, viral infection, inflammation, metabolism/endocrine disorders and neurological disorders.